Regulation of mRNA translation is a rapid and effective means to couple changes in the cellular environment with global rates of protein synthesis. An important mechanism for translational control involves GCN2 protein kinase phosphorylation of the eIF2, a translation initiator factor that delivers charged initiator tRNAs to ribosomes during translation initiation. The resulting reduction in translation initiation allows cells to conserve energy and reconfigure gene expression to remedy stress damage. Coincident with repression of global translation, phosphorylation of eIF2 (eIF2~P) directs preferential translation of specific mRNAs, such as ATF4, a transcriptional activator of genes subject to the Integrated Stress Response (ISR). Preferential translation of ATF4 during eIF2~P features two upstream ORFs (uORFs) whereby translation of the 5'-proximal uORF1 allows for reinitiating ribosomes to scan through an inhibitory uORF2 and instead translate the ATF4 coding sequence. While the ISR is critical for remediating damage accrued during acute stress, the ISR can instead switch to a terminal outcome during extended environmental challenges. Among the ISR genes, CHOP is considered to be a trigger of stress-induced apoptosis. CHOP is also subject to preferential translation during eIF2~P, although the underlying mechanism involves a single uORF that serves as a barrier to downstream translation of the CHOP coding sequence. Therefore uORFs with specific properties and arrangements in the 5'-leaders of mRNAs are central for preferential translation in the ISR. Our studies is focused on understanding the molecular mechanisms regulating protein synthesis and stress response pathways and how these processes are beneficial or adverse in a given biomedical context. This research is important for understanding the progression and treatment of many diseases, including diabetes and related metabolic disorders, cardiovascular disease, and cancer. Our hypothesis is that GCN2 recognizes different stresses and facilitates specific patterns of gene expression that are important for ameliorating cellular damage and treating and preventing disease. In this proposal, we will address how translational control triggered by eIF2~P recalibrates the protein synthetic machinery, such that mRNAs with different uORF arrangements are individually evaluated, leading to prescribed changes in translation efficiencies. Additionally, we will address the biological functions of key ISR regulators and their roles in the progression of Non-alcoholic fatty liver disease. We propose three aims. 1) Define the roles of uORFs in the differential translation of ISR regulatory genes. 2) Determine the biological function of newly identified preferentially translated genes in the ISR. 3) Determine the roles of the ISR and CHOP in hepatocyte responses to saturated free fatty acids. Completion of the proposed studies will provide critical tools for understanding and predicting translational control properties of mRNAs. These studies will also provide an understanding for how the ISR contributes to the progression of stress- related diseases, with the promise of developing new strategies for diagnosis and treatment.